Biological structures (e.g., neurons and neural networks) often extend hundreds to thousands of micrometers in three dimensions. Understanding neural computation requires the ability to measure how a neuron integrates its multitude of synaptic inputs, as well as how neural ensembles work together during sensory stimulation and motor control. With submicron lateral resolution and optical sectioning ability in scattering brains, two-photon laser scanning microscopy (2PLSM) is a powerful tool for monitoring the activity of neurons and their networks in vivo through their calcium transients. However, both individual neurons and neural networks may extend over hundreds or even thousands of micrometers in three dimensions. To view temporal dynamics of their activity (e.g., calcium transients associated with their activity) volume imaging methods with sub-second temporal resolution are needed. However, such speed is difficult to achieve with conventional two-photon laser scanning microscopy (2PLSM), because of the dependence of 2PLSM on serial focal scanning in three dimensions (3D) and the limited brightness of indicators. Because conventional 2PLSM images volume by scanning the excitation laser focus serially in 3D, the limited brightness of calcium indicators and the inertia of laser scanning units make it difficult to capture all the calcium transients in a volume at video rate (e.g., 30 Hz) while maintaining the ability to resolve synaptic structures such as dendritic spines and axonal boutons.
Thus, what is desired are techniques for high-speed, high-resolution imaging of large specimens.